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Effects of ginseng saponin administered intraperitoneally on the hypothalamo-pituitary-adrenal axis in mice
Neuroscience Letters 343 (2003) 62–66 www.elsevier.com/locate/neulet
Effects of ginseng saponin administered intraperitoneally on the hypothalamo-pituitary-adrenal axis in mice Do-Hoon Kima,1, Yoo-Sun Moonb,1, Jun-Sub Jungc, Sung-Kil Mind, Bong-Ki Sona, Hong-Won Suhc, Dong-Keun Songc,* a
b
Department of Psychiatry, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon, Kangwon-Do, 200-702, South Korea Department of Family Medicine, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon, Kangwon-Do, 200-702, South Korea c Department of Pharmacology, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon, Kangwon-Do, 200-702, South Korea d Department of Psychiatry, College of Medicine, Yonsei University, Seoul, 120-752, South Korea Received 11 November 2002; received in revised form 17 February 2003; accepted 2 March 2003
Abstract Intraperitoneal injection of ginseng total saponin (GTS; 5 and 20 mg/kg) raised plasma corticosterone levels in mice. However, interestingly, pretreatment of animals with the same doses of GTS (5 and 20 mg/kg) significantly attenuated the immobilization stressinduced increase in plasma corticosterone levels. Of the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3 injected intraperitoneally at doses of 0.1– 2 mg/kg, Rc (2 mg/kg) significantly inhibited the immobilization stress-induced increase in plasma corticosterone levels. GTS and Rc administered intraperitoneally did not affect the immobilization stress-induced elevation of plasma adrenocorticotropic hormone (ACTH) level. Pretreatment with GTS and Rc significantly attenuated the increase in plasma corticosterone levels induced by intraperitoneal injection of ACTH (30 mg/kg). These results suggest that GTS and Rc inhibit the immobilization stressinduced increase in plasma corticosterone levels by blocking ACTH action in the adrenal gland. Ginseng may be proposed to be useful for treatment of stress related disorders. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ginseng saponin; Ginsenoside Rc; Adrenocorticotropic hormone; Corticosterone; Immobilization stress
Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae) is a traditional medicine in Korea, China, and Japan. Much interest has been focused on the effects of ginseng as an adaptogen, a substance which helps the body to resist the adverse influences of a wide range of physical, chemical, and biological factors and helps the restoration of homeostasis irrespective of the direction of altered physiological function [3]. Some reports suggest that ginseng shows anti-stress activities in stressful circumstances such as footshock [20], cold [4,12] and heat [12,24]. The antistress activities of ginseng may account for its observed clinical efficacy in stress related disorders like hypertension, diabetes mellitus, peptic ulcer, depression and anxiety disorder [1,2,17,18]. It is generally accepted that hypothalamo –pituitary –adrenal (HPA) axis functions to ensure the * Corresponding author. Tel.: þ82-33-240-1653; fax: þ 82-33-240-1652. E-mail address: [email protected] (D.K. Song). 1 These authors contributed equally to this study.
body adaptation and is one of the most important systems closely related to the stress. We previously reported that ginseng total saponins (GTS) injected intracerebroventricularly inhibits the intracerebroventricular injection stress-induced plasma corticosterone levels [14]. However, the mechanism of the effect of GTS on HPA axis is not yet fully understood and it is not well known how ginseng administered peripherally acts on the HPA axis under stress. Thus, we investigated the effect of GTS and various ginsenosides injected intraperitoneally on HPA axis under immobilization stress. Male ICR mice weighing 25– 30 g, supplied by MyungJin, Inc. (Seoul, Korea), were used for all the experiments. The animals were housed five per cage in a room maintained at 22 ^ 1 8C with an alternating 12/12 h light/dark cycle. Food and water were available ad libitum. GTS and ginsenosides including Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3 were supplied by the Korea Ginseng and Tobacco Research Institute (Deajon, Korea).
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00300-8
D.-H. Kim et al. / Neuroscience Letters 343 (2003) 62–66
GTS was composed of Rb1 (16.6%), Rb2 (5.4%), Rc (4.35%), Rd (1.2%), Re (2.5%), Rf (0.55%), Rg1 (2.5%) and Rg3 (0.1%). GTS, Rb1, Rb2, Rc, Rd, Re, Rf and Rg1 were dissolved in sterile saline (0.9% NaCl). Control animals received saline. 20(S)-Rg3 and 20(R)-Rg3 were dissolved in 100% dimethylsulfoxide (DMSO) and diluted to 2% DMSO just before use. Control animals for 20(S)-Rg3 and 20(R)Rg3 received saline containing 2% DMSO, which did not affect the plasma corticosterone levels in preliminary experiments. NG-nitro-L -arginine methyl ester (L -NAME; Sigma, USA), a nitric oxide synthase (NOS) inhibitor, was dissolved in normal saline solution (0.9% NaCl). The dose of L -NAME represents the salt. The immobilization stress procedure consisted of restraint of each animal for 30 min in a 50 ml Corning tube, with the nose of the mouse at the tip of the tube. Adequate ventilation was provided by means of a hole at the tip of the tube. Thirty-minute immobilization stress was found to induce a marked increase in plasma corticosterone levels in our previous study [13]. Immediately after the completion of immobilization stress, blood (400 ml) was collected from the retro-orbital venous plexus for the assays of plasma corticosterone and ACTH. Plasma was separated by centrifugation and stored at 2 80 8C until assayed. Plasma corticosterone levels were determined by the fluorometric determination method [7]. Plasma ACTH levels were measured by radioimmunoassay as previously described [19] using a kit from Incstar (Stillwater, MN, USA). One day prior to the experiment, mice caged in groups of five were acclimatized to the condition of a quiet laboratory room overnight. Experiments were performed between 9 –11 a.m. each day to avoid the diurnal variation of plasma corticosterone levels. Each mouse was bled once and sacrificed. When a mouse was bled from the retroorbital plexus and killed, other remaining mice were separated to avoid visual and auditory stimulation. Statistical analysis was carried out by ANOVA with post-hoc Bonferroni test (Figs. 1– 3) or Student’s t-test (Fig. 4). P values less than 0.05 were considered to indicate statistical significance. To examine the effect of GTS on the HPA axis in basal state, various doses of GTS (1 –20 mg/kg) were injected intraperitoneally and blood was collected 30 min after GTS injection. As shown in Fig. 1A, GTS raised plasma corticosterone levels dose dependently in non-stressed mice. Next, to study the effect of ginseng on HPA axis during immobilization stress, various doses of GTS (1 –20 mg/kg, i.p.) were injected 15 min before the application of 30 min-immobilization stress. In contrast to the plasma corticosterone elevating effect of GTS in the basal state (Fig. 1A), GTS significantly attenuated the immobilization stress-induced plasma corticosterone levels in a dose dependent manner (Fig. 1B). To determine the active ginsenoside that can inhibit the immobilization stressinduced plasma corticosterone level, various doses of each ginsenoside (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3, 0.1-2 mg/kg, i.p.) were injected 15 min before
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Fig. 1. Effects of various doses of GTS on plasma corticosterone levels in non-stressed (A) and immobilization-stressed mice (B). Effects of various doses of ginsenoside Rc on the immobilization-stress-induced plasma corticosterone levels (C). Various doses of GTS and ginsenoside Rc were injected intraperitoneally 15 min before 30 min immobilzation stress. Blood samples were obtained immediately after completion of the procedure. For non-stressed mice, blood samples were obtained 30 min after intraperitoneal injection of GTS (20 mg/kg) or saline (control). Data are means ^ SEM (n ¼ 8 or 16). *P , 0:05, ***P , 0:001 significantly different from saline-treated control animals.
the application of 30 min-immobilization stress. Rc significantly inhibited the immobilization stress-induced plasma corticosterone level at the dose of 2 mg/kg (Fig. 1C). Rb1, Rb2, Rd, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3 were not effective (data not shown). Time-course study in which immobilization stress was applied for 0, 5, 10, 15, and 30 min showed that plasma
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Fig. 4. NG-nitro-L -arginine methyl ester (L -NAME) did not affect the ginseng total saponin (GTS) inhibition of immobilization stress-induced plasma corticosterone levels. L -NAME (10 mg/kg, i.p.) was co-injected with either saline (open columns) or GTS (20 mg/kg, i.p.) 15 min before the 30 min immobilization stress. Blood samples were obtained immediately after completion of the procedure. The data are means ^ SEM (n ¼ 8). ***P , 0:001, significantly different from saline treated control animals.
Fig. 2. (A) Effects of duration of immobilization on plasma ACTH levels. Animals were immobilized for the indicated durations (0, 5, 10, 15 and 30 min) before blood samples were obtained. (B) Effects of GTS and ginsenoside Rc on plasma ACTH levels in non-stressed and immobilization-stressed mice. GTS (20 mg/kg), Rc (2 mg/kg) or saline (control) were intraperitoneally injected 15 min before 30 min immobilzation stress. Blood samples were obtained immediately after completion of the procedure. For non-stressed mice, blood samples were obtained 30 min after intraperitoneal injection of GTS (20 mg/kg), Rc (2 mg/kg) or saline (control). The data are means ^ SEM (n ¼ 5 – 11). *P , 0:05, **P , 0:01, ***P , 0:001 significantly different from non-stressed control animals.
Fig. 3. Effects of GTS and ginsenoside Rc on the ACTH-induced increase of plasma corticosterone level. GTS (20 mg/kg) and Rc (2 mg/kg) were intraperitoneally (i.p.) administered 15 min before ACTH (30 mg/kg, i.p.) was injected. Blood samples were obtained 30 min after ACTH treatment. The data are means ^ SEM (n ¼ 5 – 8). #P , 0:05, **P , 0:01, significantly different from their respective control animals.
ACTH level rapidly increased from the baseline value, reaching a maximum at 10 min immobilization stress, then decreased moderately at 15 and 30 min immobilization stress (Fig. 2A). Next, we studied the effect of GTS and Rc on plasma ACTH levels. Fig. 2B shows that intraperitoneal injection of GTS and Rc induced a significant increase in plasma ACTH levels when measured at 30 min after the injection in non-stressed mice. However, pretreatment of animals with GTS (20 mg/kg) and Rc (2 mg/kg) 15 min before immobilization stress did not affect the immobilization stress-induced plasma ACTH levels (Fig. 2B). Next, we examined the effect of pretreatment with GTS and Rc on plasma corticosterone levels induced by intraperitoneal administration of ACTH (30 mg/kg): GTS (20 mg/kg, i.p.) or Rc (2 mg/kg, i.p.) was pretreated 15 min before ACTH injection (30 mg/kg, i.p.) and blood samples were obtained 30 min after ACTH treatment. GTS (20 mg/kg) and Rc (2 mg/kg) significantly attenuated the plasma corticosterone level induced by ACTH administration, while GTS (20 mg/kg) and Rc (2 mg/kg) per se significantly increased plasma corticosterone levels measured 30 min after injection (Fig. 3). In our previous report, GTS injected intracerebroventricularly inhibited the intracerebroventricular injection stress-induced plasma corticosterone levels [14], and the inhibitory effect of GTS was blocked by L -NAME, an inhibitor of NOS, suggesting the involvement of nitric oxide (NO). Thus, to determine the involvement of NO in the inhibitory effect of intraperitoneally administered GTS on the HPA axis, L -NAME (10 mg/kg, i.p.) and GTS (20 mg/ kg, i.p.) were co-administered 15 min before the 30 min immobilization stress. L -NAME (10 mg/kg) did not reverse the GTS inhibition of the immobilization stress-induced increase in plasma corticosterone level (Fig. 4).
D.-H. Kim et al. / Neuroscience Letters 343 (2003) 62–66
Previous studies revealed that ginseng saponin and some individual ginsenosides including Rc increase plasma ACTH and corticosterone levels in intact rats [6,9 – 11]. The increase in plasma ACTH and corticosterone levels induced by intraperitoneal injection of GTS and Rc in mice (Figs. 1A, 2B and 3) is in line with the previous reports. However, very interestingly, we found for the first time that GTS and Rc administered intraperitoneally attenuate the acute stress-induced increase in plasma corticosterone levels. It should be noted that the effects of GTS on HPA axis was contrary in the presence of the stress. In our opinion, these phenomena suggest the homeostatic effects of ginseng saponin on the regulation of HPA axis as described in other effects of ginseng [5,16,23]. But, the exact mechanisms of homeostatic effects of ginseng saponin have not been explored until now. Previously, we showed that intracerebroventricular injection of GTS, ginsenosides Rc and Rg3(S) attenuate the intracerebroventricular injection stress-induced increase in plasma corticosterone level in mice and these effects are blocked by co-administered L -NAME, an inhibitor of NOS, suggesting the involvement of NO in the brain [14]. However, in the present study, the inhibitory effect of GTS on the immobilization stress-induced increase in plasma corticosterone level was not affected by L -NAME (Fig. 4), and Rg3(S) did not affect the immobilization stressinduced increase in plasma corticosterne level. These results suggest that the inhibitory effects of GTS in the stressinduced increase in plasma corticosterone level are possibly mediated by different mechanisms according to the components of ginseng saponin, type of stress and routes of administration. Our study showed that intraperitoneally injected GTS and Rc did not affect immobilization stress-induced elevation of plasma ACTH, but GTS and Rc significantly attenuated the intraperitoneally injected ACTH-induced increase in plasma corticosterone level (Figs. 2B and 3). These results indicate that the inhibitory effects of GTS and Rc administered intraperitoneally on the immobilization stress-induced plasma corticosterone level appear to be mediated by blocking of ACTH action peripherally in the adrenal gland. However it remains to be elucidated what mechanisms at the adrenal gland level are involved in the effects of GTS on HPA axis under stress. Among nine ginsenosides tested in the present study, only Rc inhibited the stress- induced increase in plasma corticosterone level, even though other ginsenosides (Rb1, Rb2, Rd and Re) as well as Rc reportedly increase the basal plasma corticosterone levels [9,11]. Although Rc may be responsible for the GTS inhibition of stress-induced plasma corticosterone response, other ginsenosides may also be involved in the inhibitory effects of GTS on stress-induced plasma corticosterone level, because Rc is only a small component of GTS that comprises of over 30 different ginsenosides [21], of which we examined the effects of only nine ginsenosides.
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Glucocorticoid secretion serves both to alert the organism to environmental or physiologic changes and to defend homeostasis under stressful conditions. Because GTS and Rc administered intraperitoneally increase plasma corticosterone and ACTH level in basal state, it appears that GTS may serve as a mild activator of HPA axis in non-stressed state. From these results, ginseng saponin may be regarded as a kind of stressful agent whose effect is superimposed on the basal level of HPA axis function [9]. On the other hand, it has been reported that appropriately increased corticosteroids and ACTH improve selectivity, discrimination, motivation, performance, arousal and vitality [6]. Thus, it could also be insisted that an appropriate stimulation of HPA axis by ginseng saponin may help healthy people to improve their ability to deal with stressful environmental situations [6]. However, hypersecrection of glucocorticoids can promote the development of physiologic and psychological dysfunction, and inappropriate regulation of stress has been implicated in the pathogenesis of stress related disorders (for example, asthma, hypertension, colitis, depression, post-traumatic stress disorder, and dementia) [8]. Suppressive drugs on the HPA axis are recommended as a new treatment strategy for the clinical and endocrine manifestations of depression [15,22]. Thus, our results support that ginseng may be useful for treatment of such stress related disorders.
Acknowledgements This study was supported by the Hallym Academy of Sciences (2003-10-1), Hallym University, Korea.
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[9] S. Hiai, H. Yokoyama, H. Oura, Features of ginseng saponin-induced corticosterone secretion, Endocrinol. Jpn. 26 (1979) 737–740. [10] S. Hiai, H. Yokoyama, H. Oura, Y. Kawashima, Evaluation of corticosterone secretion-inducing activities of ginsenosides and their prosapogenins and sapogenins, Chem. Pharm. Bull. (Tokyo) 31 (1983) 168–174. [11] S. Hiai, H. Yokoyama, H. Oura, S. Yano, Stimulation of pituitaryadrenocortical system by ginseng saponin, Endocrinol. Jpn. 26 (1979) 661–665. [12] C. Kim, C. Kim, M. Kim, C. Hu, J. Rhe, Influence of ginseng on the stress mechanism, Lloydia 33 (1970) 43–48. [13] D.H. Kim, J.S. Jung, H.S. Kim, H.W. Suh, B.K. Son, Y.H. Kim, D.K. Song, Inhibition of brain protein kinase C attenuates immobilization stress- induced plasma corticosterone levels in mice, Neurosci. Lett. 291 (2000) 69 –72. [14] D.H. Kim, J.S. Jung, H.W. Suh, S.O. Huh, S.K. Min, B.K. Son, J.H. Park, N.D. Kim, Y.H. Kim, D.K. Song, Inhibition of stress-induced plasma corticosterone levels by ginsenosides in mice: involvement of nitric oxide, NeuroReport 9 (1998) 2261–2264. [15] B. Murphy, D. Filipini, A. Ghadirian, Possible use of glucocorticoid receptor antagonists in the treatment of major depression: preliminary results using RU 486, J. Psychiatry Neurosci. 18 (1993) 209–213. [16] S. Oh, H.S. Kim, Y.H. Seong, Effects of ginsenosides on the glutamate release and intracellular calcium level in cultured rat cerebellar neruonal cells, Arch. Pharm. Res. 18 (1995) 295–300.
[17] I. Popov, Clinical use of ginseng extract as adjuvant in revitalization therapies, in: The Central Research Institute, Office of Monopoly (Eds.), Proceedings of the International Ginseng Symposium Seoul, Korea, 1974, pp. 115. [18] I. Popov, W. Goldwag, A review of the properties and clinical effects of ginseng, Am. J. Chin. Med. 1 (1973) 263–270. [19] C. Rivier, Blockade of nitric oxide formation augments adrenocorticotropin released by blood-borne interleukin-1 beta: role of vasopressin, prostaglandins, and alpha 1-adrenergic receptors, Endocrinology 136 (1995) 3597– 3603. [20] M. Takahashi, S. Tokuyama, H. Kaneto, Anti-stress effect of ginseng on the inhibition of the development of morphine tolerance in stressed mice, Jpn. J. Pharmacol. 59 (1992) 399–404. [21] W. Tang, G. Eisenbrand, Chinese drugs of plant origin, SpringerVelag, Berlin, 1992. [22] J. Thakore, T. Dinan, Cortisol synthesis inhibition: a new treatment strategy for the clinical and endocrine manifestations of depression, Biol. Psychiatry 37 (1995) 364 –368. [23] H. Yoshimatsu, T. Sakata, H. Machidori, K. Fujimoto, A. Yamatodani, H. Wada, Ginsenoside Rg1 prevents histaminergic modulation of rat adaptive behavior from elevation of ambient temperature, Physiol. Behav. 53 (1993) 1–4. [24] W. Yuan, X. Wu, F. Yang, Effects of ginseng root saponins on brain monoamine and serum corticosterone in heat stressed mice, C.P. Association, Beijing, 1988.
Effects of ginseng saponin administered intraperitoneally on the hypothalamo-pituitary-adrenal axis in mice Do-Hoon Kima,1, Yoo-Sun Moonb,1, Jun-Sub Jungc, Sung-Kil Mind, Bong-Ki Sona, Hong-Won Suhc, Dong-Keun Songc,* a
b
Department of Psychiatry, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon, Kangwon-Do, 200-702, South Korea Department of Family Medicine, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon, Kangwon-Do, 200-702, South Korea c Department of Pharmacology, College of Medicine, Institute of Natural Medicine, Hallym University, Chunchon, Kangwon-Do, 200-702, South Korea d Department of Psychiatry, College of Medicine, Yonsei University, Seoul, 120-752, South Korea Received 11 November 2002; received in revised form 17 February 2003; accepted 2 March 2003
Abstract Intraperitoneal injection of ginseng total saponin (GTS; 5 and 20 mg/kg) raised plasma corticosterone levels in mice. However, interestingly, pretreatment of animals with the same doses of GTS (5 and 20 mg/kg) significantly attenuated the immobilization stressinduced increase in plasma corticosterone levels. Of the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3 injected intraperitoneally at doses of 0.1– 2 mg/kg, Rc (2 mg/kg) significantly inhibited the immobilization stress-induced increase in plasma corticosterone levels. GTS and Rc administered intraperitoneally did not affect the immobilization stress-induced elevation of plasma adrenocorticotropic hormone (ACTH) level. Pretreatment with GTS and Rc significantly attenuated the increase in plasma corticosterone levels induced by intraperitoneal injection of ACTH (30 mg/kg). These results suggest that GTS and Rc inhibit the immobilization stressinduced increase in plasma corticosterone levels by blocking ACTH action in the adrenal gland. Ginseng may be proposed to be useful for treatment of stress related disorders. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ginseng saponin; Ginsenoside Rc; Adrenocorticotropic hormone; Corticosterone; Immobilization stress
Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae) is a traditional medicine in Korea, China, and Japan. Much interest has been focused on the effects of ginseng as an adaptogen, a substance which helps the body to resist the adverse influences of a wide range of physical, chemical, and biological factors and helps the restoration of homeostasis irrespective of the direction of altered physiological function [3]. Some reports suggest that ginseng shows anti-stress activities in stressful circumstances such as footshock [20], cold [4,12] and heat [12,24]. The antistress activities of ginseng may account for its observed clinical efficacy in stress related disorders like hypertension, diabetes mellitus, peptic ulcer, depression and anxiety disorder [1,2,17,18]. It is generally accepted that hypothalamo –pituitary –adrenal (HPA) axis functions to ensure the * Corresponding author. Tel.: þ82-33-240-1653; fax: þ 82-33-240-1652. E-mail address: [email protected] (D.K. Song). 1 These authors contributed equally to this study.
body adaptation and is one of the most important systems closely related to the stress. We previously reported that ginseng total saponins (GTS) injected intracerebroventricularly inhibits the intracerebroventricular injection stress-induced plasma corticosterone levels [14]. However, the mechanism of the effect of GTS on HPA axis is not yet fully understood and it is not well known how ginseng administered peripherally acts on the HPA axis under stress. Thus, we investigated the effect of GTS and various ginsenosides injected intraperitoneally on HPA axis under immobilization stress. Male ICR mice weighing 25– 30 g, supplied by MyungJin, Inc. (Seoul, Korea), were used for all the experiments. The animals were housed five per cage in a room maintained at 22 ^ 1 8C with an alternating 12/12 h light/dark cycle. Food and water were available ad libitum. GTS and ginsenosides including Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3 were supplied by the Korea Ginseng and Tobacco Research Institute (Deajon, Korea).
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00300-8
D.-H. Kim et al. / Neuroscience Letters 343 (2003) 62–66
GTS was composed of Rb1 (16.6%), Rb2 (5.4%), Rc (4.35%), Rd (1.2%), Re (2.5%), Rf (0.55%), Rg1 (2.5%) and Rg3 (0.1%). GTS, Rb1, Rb2, Rc, Rd, Re, Rf and Rg1 were dissolved in sterile saline (0.9% NaCl). Control animals received saline. 20(S)-Rg3 and 20(R)-Rg3 were dissolved in 100% dimethylsulfoxide (DMSO) and diluted to 2% DMSO just before use. Control animals for 20(S)-Rg3 and 20(R)Rg3 received saline containing 2% DMSO, which did not affect the plasma corticosterone levels in preliminary experiments. NG-nitro-L -arginine methyl ester (L -NAME; Sigma, USA), a nitric oxide synthase (NOS) inhibitor, was dissolved in normal saline solution (0.9% NaCl). The dose of L -NAME represents the salt. The immobilization stress procedure consisted of restraint of each animal for 30 min in a 50 ml Corning tube, with the nose of the mouse at the tip of the tube. Adequate ventilation was provided by means of a hole at the tip of the tube. Thirty-minute immobilization stress was found to induce a marked increase in plasma corticosterone levels in our previous study [13]. Immediately after the completion of immobilization stress, blood (400 ml) was collected from the retro-orbital venous plexus for the assays of plasma corticosterone and ACTH. Plasma was separated by centrifugation and stored at 2 80 8C until assayed. Plasma corticosterone levels were determined by the fluorometric determination method [7]. Plasma ACTH levels were measured by radioimmunoassay as previously described [19] using a kit from Incstar (Stillwater, MN, USA). One day prior to the experiment, mice caged in groups of five were acclimatized to the condition of a quiet laboratory room overnight. Experiments were performed between 9 –11 a.m. each day to avoid the diurnal variation of plasma corticosterone levels. Each mouse was bled once and sacrificed. When a mouse was bled from the retroorbital plexus and killed, other remaining mice were separated to avoid visual and auditory stimulation. Statistical analysis was carried out by ANOVA with post-hoc Bonferroni test (Figs. 1– 3) or Student’s t-test (Fig. 4). P values less than 0.05 were considered to indicate statistical significance. To examine the effect of GTS on the HPA axis in basal state, various doses of GTS (1 –20 mg/kg) were injected intraperitoneally and blood was collected 30 min after GTS injection. As shown in Fig. 1A, GTS raised plasma corticosterone levels dose dependently in non-stressed mice. Next, to study the effect of ginseng on HPA axis during immobilization stress, various doses of GTS (1 –20 mg/kg, i.p.) were injected 15 min before the application of 30 min-immobilization stress. In contrast to the plasma corticosterone elevating effect of GTS in the basal state (Fig. 1A), GTS significantly attenuated the immobilization stress-induced plasma corticosterone levels in a dose dependent manner (Fig. 1B). To determine the active ginsenoside that can inhibit the immobilization stressinduced plasma corticosterone level, various doses of each ginsenoside (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3, 0.1-2 mg/kg, i.p.) were injected 15 min before
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Fig. 1. Effects of various doses of GTS on plasma corticosterone levels in non-stressed (A) and immobilization-stressed mice (B). Effects of various doses of ginsenoside Rc on the immobilization-stress-induced plasma corticosterone levels (C). Various doses of GTS and ginsenoside Rc were injected intraperitoneally 15 min before 30 min immobilzation stress. Blood samples were obtained immediately after completion of the procedure. For non-stressed mice, blood samples were obtained 30 min after intraperitoneal injection of GTS (20 mg/kg) or saline (control). Data are means ^ SEM (n ¼ 8 or 16). *P , 0:05, ***P , 0:001 significantly different from saline-treated control animals.
the application of 30 min-immobilization stress. Rc significantly inhibited the immobilization stress-induced plasma corticosterone level at the dose of 2 mg/kg (Fig. 1C). Rb1, Rb2, Rd, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3 were not effective (data not shown). Time-course study in which immobilization stress was applied for 0, 5, 10, 15, and 30 min showed that plasma
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D.-H. Kim et al. / Neuroscience Letters 343 (2003) 62–66
Fig. 4. NG-nitro-L -arginine methyl ester (L -NAME) did not affect the ginseng total saponin (GTS) inhibition of immobilization stress-induced plasma corticosterone levels. L -NAME (10 mg/kg, i.p.) was co-injected with either saline (open columns) or GTS (20 mg/kg, i.p.) 15 min before the 30 min immobilization stress. Blood samples were obtained immediately after completion of the procedure. The data are means ^ SEM (n ¼ 8). ***P , 0:001, significantly different from saline treated control animals.
Fig. 2. (A) Effects of duration of immobilization on plasma ACTH levels. Animals were immobilized for the indicated durations (0, 5, 10, 15 and 30 min) before blood samples were obtained. (B) Effects of GTS and ginsenoside Rc on plasma ACTH levels in non-stressed and immobilization-stressed mice. GTS (20 mg/kg), Rc (2 mg/kg) or saline (control) were intraperitoneally injected 15 min before 30 min immobilzation stress. Blood samples were obtained immediately after completion of the procedure. For non-stressed mice, blood samples were obtained 30 min after intraperitoneal injection of GTS (20 mg/kg), Rc (2 mg/kg) or saline (control). The data are means ^ SEM (n ¼ 5 – 11). *P , 0:05, **P , 0:01, ***P , 0:001 significantly different from non-stressed control animals.
Fig. 3. Effects of GTS and ginsenoside Rc on the ACTH-induced increase of plasma corticosterone level. GTS (20 mg/kg) and Rc (2 mg/kg) were intraperitoneally (i.p.) administered 15 min before ACTH (30 mg/kg, i.p.) was injected. Blood samples were obtained 30 min after ACTH treatment. The data are means ^ SEM (n ¼ 5 – 8). #P , 0:05, **P , 0:01, significantly different from their respective control animals.
ACTH level rapidly increased from the baseline value, reaching a maximum at 10 min immobilization stress, then decreased moderately at 15 and 30 min immobilization stress (Fig. 2A). Next, we studied the effect of GTS and Rc on plasma ACTH levels. Fig. 2B shows that intraperitoneal injection of GTS and Rc induced a significant increase in plasma ACTH levels when measured at 30 min after the injection in non-stressed mice. However, pretreatment of animals with GTS (20 mg/kg) and Rc (2 mg/kg) 15 min before immobilization stress did not affect the immobilization stress-induced plasma ACTH levels (Fig. 2B). Next, we examined the effect of pretreatment with GTS and Rc on plasma corticosterone levels induced by intraperitoneal administration of ACTH (30 mg/kg): GTS (20 mg/kg, i.p.) or Rc (2 mg/kg, i.p.) was pretreated 15 min before ACTH injection (30 mg/kg, i.p.) and blood samples were obtained 30 min after ACTH treatment. GTS (20 mg/kg) and Rc (2 mg/kg) significantly attenuated the plasma corticosterone level induced by ACTH administration, while GTS (20 mg/kg) and Rc (2 mg/kg) per se significantly increased plasma corticosterone levels measured 30 min after injection (Fig. 3). In our previous report, GTS injected intracerebroventricularly inhibited the intracerebroventricular injection stress-induced plasma corticosterone levels [14], and the inhibitory effect of GTS was blocked by L -NAME, an inhibitor of NOS, suggesting the involvement of nitric oxide (NO). Thus, to determine the involvement of NO in the inhibitory effect of intraperitoneally administered GTS on the HPA axis, L -NAME (10 mg/kg, i.p.) and GTS (20 mg/ kg, i.p.) were co-administered 15 min before the 30 min immobilization stress. L -NAME (10 mg/kg) did not reverse the GTS inhibition of the immobilization stress-induced increase in plasma corticosterone level (Fig. 4).
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Previous studies revealed that ginseng saponin and some individual ginsenosides including Rc increase plasma ACTH and corticosterone levels in intact rats [6,9 – 11]. The increase in plasma ACTH and corticosterone levels induced by intraperitoneal injection of GTS and Rc in mice (Figs. 1A, 2B and 3) is in line with the previous reports. However, very interestingly, we found for the first time that GTS and Rc administered intraperitoneally attenuate the acute stress-induced increase in plasma corticosterone levels. It should be noted that the effects of GTS on HPA axis was contrary in the presence of the stress. In our opinion, these phenomena suggest the homeostatic effects of ginseng saponin on the regulation of HPA axis as described in other effects of ginseng [5,16,23]. But, the exact mechanisms of homeostatic effects of ginseng saponin have not been explored until now. Previously, we showed that intracerebroventricular injection of GTS, ginsenosides Rc and Rg3(S) attenuate the intracerebroventricular injection stress-induced increase in plasma corticosterone level in mice and these effects are blocked by co-administered L -NAME, an inhibitor of NOS, suggesting the involvement of NO in the brain [14]. However, in the present study, the inhibitory effect of GTS on the immobilization stress-induced increase in plasma corticosterone level was not affected by L -NAME (Fig. 4), and Rg3(S) did not affect the immobilization stressinduced increase in plasma corticosterne level. These results suggest that the inhibitory effects of GTS in the stressinduced increase in plasma corticosterone level are possibly mediated by different mechanisms according to the components of ginseng saponin, type of stress and routes of administration. Our study showed that intraperitoneally injected GTS and Rc did not affect immobilization stress-induced elevation of plasma ACTH, but GTS and Rc significantly attenuated the intraperitoneally injected ACTH-induced increase in plasma corticosterone level (Figs. 2B and 3). These results indicate that the inhibitory effects of GTS and Rc administered intraperitoneally on the immobilization stress-induced plasma corticosterone level appear to be mediated by blocking of ACTH action peripherally in the adrenal gland. However it remains to be elucidated what mechanisms at the adrenal gland level are involved in the effects of GTS on HPA axis under stress. Among nine ginsenosides tested in the present study, only Rc inhibited the stress- induced increase in plasma corticosterone level, even though other ginsenosides (Rb1, Rb2, Rd and Re) as well as Rc reportedly increase the basal plasma corticosterone levels [9,11]. Although Rc may be responsible for the GTS inhibition of stress-induced plasma corticosterone response, other ginsenosides may also be involved in the inhibitory effects of GTS on stress-induced plasma corticosterone level, because Rc is only a small component of GTS that comprises of over 30 different ginsenosides [21], of which we examined the effects of only nine ginsenosides.
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Glucocorticoid secretion serves both to alert the organism to environmental or physiologic changes and to defend homeostasis under stressful conditions. Because GTS and Rc administered intraperitoneally increase plasma corticosterone and ACTH level in basal state, it appears that GTS may serve as a mild activator of HPA axis in non-stressed state. From these results, ginseng saponin may be regarded as a kind of stressful agent whose effect is superimposed on the basal level of HPA axis function [9]. On the other hand, it has been reported that appropriately increased corticosteroids and ACTH improve selectivity, discrimination, motivation, performance, arousal and vitality [6]. Thus, it could also be insisted that an appropriate stimulation of HPA axis by ginseng saponin may help healthy people to improve their ability to deal with stressful environmental situations [6]. However, hypersecrection of glucocorticoids can promote the development of physiologic and psychological dysfunction, and inappropriate regulation of stress has been implicated in the pathogenesis of stress related disorders (for example, asthma, hypertension, colitis, depression, post-traumatic stress disorder, and dementia) [8]. Suppressive drugs on the HPA axis are recommended as a new treatment strategy for the clinical and endocrine manifestations of depression [15,22]. Thus, our results support that ginseng may be useful for treatment of such stress related disorders.
Acknowledgements This study was supported by the Hallym Academy of Sciences (2003-10-1), Hallym University, Korea.
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